In the global fight against infectious disease threats—from SARS-CoV-2 and influenza to drug-resistant bacteria—rapid, sensitive, and field-ready diagnostic tools are urgently needed. A newly published study in Pathogens offers a comprehensive review of emerging nucleic acid detection technologies powered by Argonaute (Ago) proteins. Conducted by researchers at the First Affiliated Hospital of Zhejiang University School of Medicine and associated national research centers in China, the paper underscores the growing relevance of Ago-based diagnostics in precision public health and biosurveillance.
Why Argonaute? A New Class of Programmable Nucleases
Argonaute proteins are programmable nucleases naturally involved in RNA interference and gene silencing. Unlike CRISPR/Cas systems, which require specific recognition motifs (PAM sites), Ago proteins can target and cleave DNA or RNA without such constraints. This versatility, along with their ability to detect single-nucleotide variants and support multiplexing, positions Ago-based tools as a promising next-generation solution for molecular diagnostics—especially in low-resource or point-of-care settings.
Advances in Ago-Powered Detection Platforms
The study catalogs a wide range of Argonaute-based diagnostic systems developed in recent years, highlighting key technological innovations:
- PAND (PfAgo-based): Uses a thermophilic Argonaute to cleave target DNA and amplify signal via fluorescent probes, achieving attomolar sensitivity.
- A-Star: Enables precise single-nucleotide variant detection through a clever “one-tube” PCR approach, combining wild-type cleavage with selective mutant amplification.
- USPCRP: Integrates ultra-short primers with PfAgo for streamlined, highly specific DNA cleavage and detection using minimal reagents.
- TEAM: Merges TtAgo’s specificity with exponential isothermal amplification to detect microRNA with single-molecule sensitivity.
- MULAN: Offers rapid, portable multiplex detection of viruses such as SARS-CoV-2, influenza A/B, and HPV with a total reaction time under 45 minutes.
- PASS: An amplification-free detection platform that pairs Argonaute cleavage with deep-learning-based signal quantification.
Each system leverages unique biochemical features of Ago proteins—such as DNA-guided cleavage, thermal stability, and compatibility with fluorescence reporting—to improve detection speed, cost-efficiency, and diagnostic breadth.
Real-World Applications in Pathogen Detection
The authors detail how these technologies are being applied to high-priority public health targets:
Bacterial Infections
- Detection of multidrug-resistant Klebsiella pneumoniae directly from blood and urine using the ANCA system.
- Identification of Salmonella typhimurium with CbAgo-powered biosensors, offering detection limits as low as 40.5 CFU/mL.
- Dual-gene detection of methicillin-resistant Staphylococcus aureus using the STAND system.
Viral Infections
- Differentiation of Influenza A/B and SARS-CoV-2 using the portable MULAN system.
- Subtype-specific detection of human papillomavirus (HPV) and SARS-CoV-2 variants via PfAgo systems.
- Use of Ago-GFET nanoplatforms to discriminate single-base viral RNA mutations with >90% accuracy.
Fungal and Food Safety Testing
- Detection of foodborne mycotoxins like aflatoxin B1 and zearalenone using microsphere encoding platforms guided by CbAgo.
- Multi-target detection systems designed for food safety and agricultural monitoring.
Mycoplasma and Aquaculture Pathogens
- On-site detection of Mycoplasma synoviae in poultry with a sensitivity of just 2 copies/mL.
- Deployment of Ago-based diagnostics to detect Enterocytozoon hepatopenaei (EHP) in shrimp farming operations, supporting early and precise disease control.
Strengths and Challenges of Ago-Based Technologies
The review emphasizes the technical advantages of Ago-based diagnostics:
- High specificity down to single-nucleotide mutations.
- PAM-independent targeting, allowing greater design flexibility.
- Low-cost, stable guide DNAs suitable for field deployment.
- Potential for multiplexing and rapid turnaround (often <30 minutes).
However, the authors also caution that several hurdles remain:
- Systems often require multiple steps and guide sequences, raising complexity.
- Thermostability of commonly used Agos limits some platforms to high-temperature operations.
- Further research is needed to optimize signal output modalities beyond fluorescence and reduce false positives due to nonspecific Apo protein activity.
Future Directions: From Lab to Field
The study calls for integrating Argonaute diagnostics with emerging technologies such as microfluidics, artificial intelligence-guided probe design, and multimodal sensing platforms (e.g., electrochemical, Raman spectroscopy). Researchers envision a future of self-contained, amplification-free detection kits for decentralized testing in clinics, homes, farms, and remote outbreak zones.
With continued refinement and smart engineering, Ago-based detection systems could support the rapid response and real-time surveillance capabilities demanded by today’s global health landscape.
Hong, M., Wu, G., Ren, Y., Wu, S., Zhu, H., & Chen, Z. (2025). Advancements in Pathogen Detection: Argonaute-Based Nucleic Acid Detection Technology. Pathogens, 14(6), 554. https://doi.org/10.3390/pathogens14060554